International Patent Application No WO91/13167 (applicant, The Board of Trustees of Leyland Stanford University, and hereinafter referred to as WO91/13167) describes the identification, characterization, expression and uses of insect steroid receptors and DNA sequences encoding same and, in particular, the identification, characterization, expression and uses of the steroid receptor of the common fruit fly, Drosophila melanogaster. 
It has been found by the present inventors that the limited homology between the D. melanogaster steroid receptor-encoding gene sequences and the steroid receptor-encoding sequences derived from other insects, in particular those derived from diptera such as the Australian sheep blowfly L. cuprina, hemiptera such as aphids, scale insects and leaf hoppers, coleoptera, neuroptera, lepidoptera, and ants, as well as from helminths and protozoa, prevents the routine isolation of DNA sequences encoding steroid receptors and/or juvenile hormone receptors from these latter-mentioned organisms.
Moreover, the present inventors have discovered that the D. melanogaster steroid receptor described in WO91/13167 is temperature-sensitive, showing reduced activity at temperatures above 30° C., such as at temperatures about 37° C., particularly at low concentrations of the receptor. Accordingly, the D. melanogaster steroid receptor described in WO91/13167 is of limited utility at physiological temperatures applicable to animal or bacterial cells. Moreover, wherein it is desirable to produce a biologically-active steroid receptor using in vivo or in situ expression systems, which expression systems routinely utilise cells or tissues in the temperature range of about 28° C. to about 42° C., the D. melanogaster steroid receptor is also of limited utility.
In work leading up to the present invention, the present inventors developed a novel screening protocol, involving the utilisation of highly-degenerate oligonucleotide probes and primers derived from the amino acid sequences of the DNA-binding domains of the D. melanogaster and Chironomus tentans ecdysone receptor polypeptides, to identify nucleotide sequences encoding novel steroid receptor polypeptides and novel insect juvenile hormone receptor polypeptides. The present inventors have further identified specific regions within these novel polypeptides which are suitable for use in preparing a surprising range of novel steroid receptor polypeptide derivatives and insect juvenile hormone receptor polypeptide derivatives. The novel steroid receptor polypeptides and novel insect juvenile hormone receptor polypeptides of the present invention, and derivative polypeptides thereof, and assembled steroid receptors and insect juvenile hormone receptors derived from said polypeptides and derivatives, and nucleic acid molecules encoding same as exemplified herein, provide the means for developing a wide range of insecticidally-active agents, as well as methods for the regulated production of bioactive molecules. In particular, the present invention provides the means for developing specific ligands which bind to and either agonise or antagonise the steroid receptors and/or juvenile hormone receptors and/or polypeptide subunits thereof as described herein, thereby functioning as highly-specific insecticides, offering significant commercial and environmental benefits.
The present inventors have been surprisingly successful in characterizing the ecdysone receptor and juvenile hormone receptor derived from insects of the orders Diptera and Hemiptera, and polypeptide components thereof and functional derivatives of said polypeptides and receptors, particularly in light of the extreme difficulties in dealing with these organisms. The nature of these molecules was unknown prior to the present invention.
The various aspects of this invention overcome the problems associated with Drosophila ecdysone receptors which lack thermal stability. Moreover, those aspects of the invention pertaining to methods of screening for insecticidally active agents do not involve competition assays which are generally complex, and often inaccurate or difficult to calibrate.